The overall goal of the proposed experiments is to dissect the cellular and biochemical requirements for the transition of CD4+CD8+ thymocytes to mature CD4+ thymocytes. The investigator has developed the only in vitro model for positive selection using cloned cell lines as both stimulators and responders. In this case, the responding cell population is DPK, a CD4+CD8+ line derived from a spontaneous thymoma from an AND TCR transgenic mouse. This TCR is specific for pigeon cytochrome c and the class II molecule I-E of the b, s, or k haplotypes. The cloned stimulator cell line is IMBR3, a conditionally immortalized cortical epithelial cell line from an I-Ek thymus. Specific Aim 1 will examine the costimulatory and antigen presentation properties of the cortical TE cell line IMBR3. The goal of Specific Aim 2 is the identification of cell surface molecules involved in the function of cortical TE cell lines. Antibodies will be generated against IMBR3 and screened on trypsinized versus dispase-treated IMBR3 cells and for blocking of the IMBR3-induced changes in DPK cells. Attempts will also be made to identify the trypsin-sensitive element of IMBR3 cells by analysis of supernatants from biotinylated, trypsinized IMBR3 cells. A third approach, if necessary, will involve subtractive hybridization to clone genes characterized by differential expression between IMBR3 and subcloned IMBR3 cells that lack inductive properties relative to their parent line. These subclones have already been derived and their activity compared to that of the parent line has been well documented. Specific Aim 3 is designed to analyze the requirements for the induction of the transition from double positive to single positive thymocyte, along the CD4 lineage. The final aim is directed at determining whether single, multiple, or sustained TCR engagements are required for positive selection.